Novel agents and uses thereof

ABSTRACT

The present invention provides agents comprising or consisting of a binding moiety with specificity for interleukin-1 receptor accessory protein (IL 1 RAP) for use in inducing cell death and/or inhibiting the growth and/or proliferation of pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express IL 1 RAP. A related aspect of the invention provides agents comprising or consisting of a binding moiety with specificity for interleukin-1 receptor accessory protein (IL 1 RAP) for use in detecting pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express IL 1 RAP. Further provided are pharmacological compositions comprising the agents of the invention and methods of using the same.

FIELD OF INVENTION

The present invention relates to agents for use in the treatment anddiagnosis of neoplastic hematologic disorders, such as chronic myeloidleukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplasticsyndrome (MDS), myeloproliferative disorders (MPD) and acute myeloidleukemia (AML).

BACKGROUND

Chronic myeloid leukemia (CML) was the first human neoplasm associatedwith a recurrent genetic aberration; the Philadelphia (Ph) chromosome,formed through a reciprocal translocation between chromosome 9 and 22,giving rise to the constitutively active tyrosine kinase BCR/ABL1¹. InCML, the Ph chromosome is believed to originate in a hematopoietic stemcell (HSC) as it clonally can be found both in malignant myeloid cellsand non-malignant lymphoid cells². The Ph chromosome is also found in afraction of acute lymphoblastic leukemia (ALL) and acute myeloidleukemia (AML). CML is comprised of heterogeneous cell types of variousmaturation stages that are maintained by a small number of cells, termedCML stem cells, sharing the capacity to self-renew with normal HSC³. Ithas been demonstrated that the CML stem cells are at least partiallyresistant to current treatments with tyrosine kinase inhibitors^(4, 5),which despite clinical success show a suppressive rather than curativeeffect in this disorder. Thus, identifying a strategy to efficientlytarget CML stem cells is highly desirable to achieve a permanent cure ofthe disorder. Such a strategy would be to identify a target on CML stemcells that may provide novel means to eradicate the CML stem cells.Encouraging reports in this direction have been described in the relateddisorder acute myeloid leukemia (AML), where antibodies targeting CD123,CXCR4, CD44 or CD47 on AML stem cells show anti-leukemic effects in AMLanimal models⁶⁻⁹. Further, AML stem cell associated antigens such asCD96 and CLL-1 have been identified^(10, 11), providing additionaltarget candidates in this disorder. Intriguingly, despite being one ofthe most studied neoplasms of all time, referred to as a stem cellcancer disorder, no cell surface biomarker has so far been identified inCML that allows a prospective separation of CML stem cells from normalHSCs, both residing in the rare CD34⁺CD38⁻ cell population^(12, 13).Identification of such a biomarker would be instrumental in thecharacterization of the CML stem cell, but could also be used for noveltreatment developments and for tracking therapeutic effects on primitiveCML cells during treatment.

Accordingly, the present invention seeks to provide agents for use inthe treatment and diagnosis of neoplastic hematologic disorders, such asCML. In addition, the invention seeks to provide agent for use intreatment and diagnosis of other neoplastic hematologic disorders, suchas ALL, AML, Ph chromosome-negative myeloproliferative disorders (MPD),and myelodysplastic syndromes (MDS).

SUMMARY OF INVENTION

The invention provides agents for use in the treatment and/or diagnosisof neoplastic hematologic disorders and evolved directly from thediscovery by the inventors that stem cells and progenitor cellsassociated with neoplastic hematologic disorders, such as chronicmyeloid leukemia, which express IL1RAP (also known as IL1-RAP) on theirsurface. In contrast, normal healthy hematopoietic stem cells (as wellas progenitor cells) do not express, or show very low expression levels,of IL1RAP. Moreover, the inventors have discovered that the stem andprogenitor cells of other neoplastic hematologic disorders, such as ALL,AML, Ph chromosome-negative myeloproliferative disorders (MPD), andmyelodysplastic syndromes (MDS), are also associated with anupregulation of IL1RAP on their cell surface. Thus, the inventionprovides agents for use in the treatment and/or diagnosis of neoplastichematologic disorders associated with upregulation of IL1RAP on thesurface of stem cells and/or progenitor cells.

A first aspect of the invention provides an agent comprising orconsisting of a binding moiety with specificity for interleukin-1receptor accessory protein (IL1RAP) for use in inducing cell death(either directly or indirectly via triggering of the immune system)and/or inhibiting the growth (i.e. size) and/or proliferation (i.e.number) of pathological stem cells and/or progenitor cells associatedwith a neoplastic hematologic disorder, wherein the stem cells and/orprogenitor cells express IL1RAP. Thus, the agent may be for use ininhibiting the growth and/or proliferation of pathological stem cellsalone, of progenitor cells alone, or of both pathological stem cells andprogenitor cells.

The agent may also be for use in inducing differentiation ofpathological stem and/or progenitor cells which express IL1RAP.

A second, related aspect of the invention provides an agent comprisingor consisting of a binding moiety with specificity for interleukin-1receptor accessory protein (IL1RAP) for use in detecting pathologicalstem cells and/or progenitor cells associated with a neoplastichematologic disorder, wherein the stem cells and/or progenitor cellsexpress IL1RAP. Thus, the agent may be for use in detecting pathologicalstem cells alone, progenitor cells alone, or both pathological stemcells and progenitor cells.

By “interleukin-1 receptor accessory protein”, “IL1RAP” and “IL1-RAP” wespecifically include the human IL1RAP protein, for example as describedin GenBank Accession No. AAB84059, NCBI Reference Sequence: NP_002173.1and UniProtKB/Swiss-Prot Accession No. Q9NPH3-1 (see also Huang et al.,1997, Proc. Natl. Acad. Sci. USA. 94 (24), 12829-12832). IL1RAP is alsoknown in the scientific literature as IL1R3, C3orf13, FLJ37788, IL-1RAcPand EG3556

By “binding moiety” we include all types of chemical entity (forexample, oligonucleotides, polynucleotide, polypeptides, peptidomimeticsand small compounds) which are capable of binding to IL1RAP.Advantageously, the binding moiety is capable of binding selectively(i.e. preferentially) to IL1RAP under physiological conditions. Thebinding moiety preferably has specificity for human IL1RAP, which may belocalised on the surface of a cell (e.g. the pathological stem cell orprogcnitor cell).

By “pathological stem cells associated with a neoplastic hematologicdisorder” we include stem cells which are responsible for thedevelopment of a neoplastic hematologic disorder in an individual, i.e.neoplastic stem cells. In particular, the pathological stem cells may beleukemic stem cells (for example, as described in Guo et al., 2008,Nature 453(7194):529-33). Such stem cell may be distinguished fromnormal hematopoietic stem cells by their expression of the cell surfaceprotein, IL1RAP (see Example below). In one embodiment, the pathologicalstem cells are CD34⁺, CD38⁻ cells

By “progenitor cells” associated with a neoplastic hematologic disorderwe include cells derived from pathological stem cells which areresponsible for the development of a neoplastic hematologic disorder inan individual. In particular, the progenitor cells may be leukemicprogenitor cells (for example, as described in Examples below; see FIG.2b ). Such progenitor cells may be distinguished from normalhematopoietic progenitor cells by their higher expression of the cellsurface protein, IL1RAP (see Example below). In one embodiment, thepathological progenitor cells are CD34⁺, CD38⁺ cells.

By “neoplastic hematologic disorder” we specifically include hematologiccancers such as leukemias, as well as leukemia-like diseases such asmyeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS).

Thus, in one embodiment of the first aspect of the invention, theneoplastic hematologic disorder is a leukemic disease or disorder, i.e.a cancer of the blood or bone marrow, which may be acute or chronic.

In a further embodiment, the neoplastic hematologic disorder may beassociated with cells comprising a BCR/ABL1 fusion gene. For example,the pathological stem cells and/or progenitor cells may comprise aBCR/ABL1 fusion gene.

In a related embodiment, the neoplastic hematologic disorder may beassociated with cells comprising a Philadelphia (Ph) chromosome. Forexample, the pathological stem cells and/or progenitor cells maycomprise a Ph chromosome. By “Ph chromosome” in this context we mean aspecific chromosomal abnormality resulting from a reciprocaltranslocation between chromosome 9 and 22, specifically designatedt(9;22)(q34;q11). An example of a neoplastic hematologic disorderassociated with cells comprising a Ph chromosome is chronic myeloid, ormyelogenous, leukemia (CML).

However, it will be appreciated by persons skilled in the art that theagents of the invention may also be used in the treatment and/ordiagnosis of neoplastic hematologic disorders which are not associatedwith cells comprising a Philadelphia (Ph) chromosome (but neverthelessshow upregulation of IL1RAP). Such neoplastic hematologic disorderswhich are associated with cells which do not comprise a Ph chromosomeinclude the myelodysplastic syndromes (MDS) and myeloproliferativedisorders (MPD) such as polycythemia vera (PV), essential thrombocytosis(ET) and myelofibrosis (MF).

More specifically, the neoplastic hematologic disorder may be selectedfrom the group consisting of chronic myeloid leukemia (CML),myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS),acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

In one particularly preferred embodiment, the neoplastic hematologicdisorder is chronic myeloid leukemia (CML).

In relation to the diagnostic aspects of the invention, it is sufficientthat the agent is merely capable of binding to IL1RAP present on thesurface of the pathological stem cells and/or progenitor cells (withouthaving any functional impact upon those cells).

In relation to the therapeutic and prophylactic aspects of theinvention, it will be appreciated by persons skilled in the art thatbinding of the agent to IL1RAP present on the surface of thepathological stem cells and/or progenitor cells may lead to a modulation(i.e. an increase or decrease) of a biological activity of IL1RAP.However, such a modulatory effect is not essential; for example, theagents of the invention may elicit a therapeutic and prophylactic effectsimply by virtue of binding to IL1RAP on the surface of the pathologicalstem cells and/or progenitor cells, which in turn may trigger the immunesystem to induce cell death (e.g. by ADCC).

By “biological activity of IL1RAP” we include any interaction orsignalling event which involves IL1RAP on pathological stem cells and/orprogenitor cells. For example, in one embodiment the agent is capable ofblocking binding of one or more co-receptors to IL1RAP (such as IL1R1,ST2, C-KIT and/or IL1RL2).

Such inhibition of the biological activity of IL1RAP by an agent of theinvention may be in whole or in part. For example, the agent may inhibitthe biological activity of IL1RAP by at least 10%, preferably at least20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and most preferably by 100%compared to the biological activity of IL1RAP in pathological stem cellsand/or progenitor cells which have not been exposed to the agent. In apreferred embodiment, the agent is capable of inhibiting the biologicalactivity of IL1RAP by 50% or more compared to the biological activity ofIL1RAP in pathological stem cells and/or progenitor cells which have notbeen exposed to the agent.

Likewise, it will be appreciated that inhibition of growth and/orproliferation of the pathological stem cells and/or progenitor cells maybe in whole or in part. For example, the agent may inhibit the growthand/or proliferation of the pathological stem cells and/or progenitorcells by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%,80% or 90%, and most preferably by 100% compared to the growth and/orproliferation of the pathological stem cells and/or progenitor cellswhich have not been exposed to the agent.

Similarly, it will be appreciated that the induction of differentiationof pathological stem cells and/or progenitor cells may be to any extent.For example, the agent may induce differentiation of the pathologicalstem cells and/or progenitor cells by at least 10%, preferably at least20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and most preferably by 100%compared to the differentiation of the pathological stem cells and/orprogenitor cells which have not been exposed to the agent.

In a further preferred embodiment, the agent is capable of killing thepathological stem cells and/or progenitor cells. In particular, theagent may be capable of inducing stem cell and/or progenitor cell deathby apoptosis or autophagy. For example, the agent may induce apoptosisby antibody-dependent cell-mediated cytotoxicity (ADCC).

As indicated above, the agents of the invention may comprise or consistof any suitable chemical entity constituting a binding moiety withspecificity for IL1RAP.

Methods for detecting interactions between a test chemical entity andIL1RAP are well known in the art. For example ultrafiltration with ionspray mass spectroscopy/HPLC methods or other physical and analyticalmethods may be used. In addition, Fluorescence Energy Resonance Transfer(FRET) methods may be used, in which binding of two fluorescent labelledentities may be measured by measuring the interaction of the fluorescentlabels when in close proximity to each other.

Alternative methods of detecting binding of IL1RAP to macromolecules,for example DNA, RNA, proteins and phospholipids, include a surfaceplasmon resonance assay, for example as described in Plant et al., 1995,Analyt Biochem 226(2), 342-348. Such methods may make use of apolypeptide that is labelled, for example with a radioactive orfluorescent label.

A further method of identifying a chemical entity that is capable ofbinding to IL1RAP is one where the protein is exposed to the compoundand any binding of the compound to the said protein is detected and/ormeasured. The binding constant for the binding of the compound to thepolypeptide may be determined. Suitable methods for detecting and/ormeasuring (quantifying) the binding of a compound to a polypeptide arewell known to those skilled in the art and may be performed, forexample, using a method capable of high throughput operation, forexample a chip-based method. New technology, called VLSIPS™, has enabledthe production of extremely small chips that contain hundreds ofthousands or more of different molecular probes. These biological chipshave probes arranged in arrays, each probe assigned a specific location.Biological chips have been produced in which each location has a scaleof, for example, ten microns. The chips can be used to determine whethertarget molecules interact with any of the probes on the chip. Afterexposing the array to target molecules under selected test conditions,scanning devices can examine each location in the array and determinewhether a target molecule has interacted with the probe at thatlocation.

Another method of identifying compounds with binding affinity for IL1RAPis the yeast two-hybrid system, where the polypeptides of the inventioncan be used to “capture” proteins that bind IL1RAP. The yeast two-hybridsystem is described in Fields & Song, Nature 340:245-246 (1989).

In one preferred embodiment, the agent comprises or consists of apolypeptide.

For example, the agent may comprise or consist of an antibody or anantigen-binding fragment thereof with binding specificity for IL1RAP, ora variant, fusion or derivative of said antibody or antigen-bindingfragment, or a fusion of a said variant or derivative thereof, whichretains the binding specificity for IL1RAP.

By “antibody” we include substantially intact antibody molecules, aswell as chimaeric antibodies, humanised antibodies, human antibodies(wherein at least one amino acid is mutated relative to the naturallyoccurring human antibodies), single chain antibodies, bispecificantibodies, antibody heavy chains, antibody light chains, homodimers andheterodimers of antibody heavy and/or light chains, and antigen bindingfragments and derivatives of the same.

By “antigen-binding fragment” we mean a functional fragment of anantibody that is capable of binding to IL1RAP.

Preferably, the antigen-binding fragment is selected from the groupconsisting of Fv fragments (e.g. single chain Fv and disulphide-bondedFv), Fab-like fragments (e.g. Fab fragments, Fab′ fragments and F(ab)₂fragments), single variable domains (e.g. V_(H) and V_(L) domains) anddomain antibodies (dAbs, including single and dual formats [i.e.dAb-linker-dAb]).

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFvand dAb antibody fragments can be expressed in and secreted from E.coli, thus allowing the facile production of large amounts of the saidfragments.

Also included within the scope of the invention are modified versions ofantibodies and antigen-binding fragments thereof, e.g. modified by thecovalent attachment of polyethylene glycol or other suitable polymer(see below).

Methods of generating antibodies and antibody fragments are well knownin the art. For example, antibodies may be generated via any one ofseveral methods which employ induction of in vivo production of antibodymolecules, screening of immunoglobulin libraries (Orlandi. et al, 1989.Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al., 1991, Nature349:293-299) or generation of monoclonal antibody molecules by celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the Epstein-Barrvirus (EBV)-hybridoma technique (Kohler et al., 1975. Nature256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81:31-42; Cote etal., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984.Mol. Cell. Biol. 62:109-120).

Suitable monoclonal antibodies to selected antigens may be prepared byknown techniques, for example those disclosed in “Monoclonal Antibodies:A manual of techniques”, H Zola (CRC Press, 1988) and in “MonoclonalHybridoma Antibodies: Techniques and Applications”, J G R Hurrell (CRCPress, 1982).

Likewise, antibody fragments can be obtained using methods well known inthe art (see, for example, Harlow & Lane, 1988, “Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory, New York). Forexample, antibody fragments according to the present invention can beprepared by proteolytic hydrolysis of the antibody or by expression inE. coli or mammalian cells (e.g. Chinese hamster ovary cell culture orother protein expression systems) of DNA encoding the fragment.Alternatively, antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies by conventional methods.

It will be appreciated by persons skilled in the art that for humantherapy or diagnostics, human or humanised antibodies are preferablyused. Humanised forms of non-human (e.g. murine) antibodies aregenetically engineered chimaeric antibodies or antibody fragments havingpreferably minimal-portions derived from non-human antibodies. Humanisedantibodies include antibodies in which complementary determining regionsof a human antibody (recipient antibody) are replaced by residues from acomplementary determining region of a non human species (donor antibody)such as mouse, rat of rabbit having the desired functionality. In someinstances, Fv framework residues of the human antibody are replaced bycorresponding non-human residues. Humanised antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported complementarity determining region or framework sequences. Ingeneral, the humanised antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the complementarity determining regions correspondto those of a non human antibody and all, or substantially all, of theframework regions correspond to those of a relevant human consensussequence. Humanised antibodies optimally also include at least a portionof an antibody constant region, such as an Fc region, typically derivedfrom a human antibody (see, for example, Jones et al., 1986. Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992,Curr. Op. Struct. Biol. 2:593-596).

Methods for humanising non-human antibodies are well known in the art.Generally, the humanised antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues, often referred to as imported residues, aretypically taken from an imported variable domain. Humanisation can beessentially performed as described (see, for example, Jones et al.,1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327;Verhoeyen et al., 1988, Science 239:1534-1536I; U.S. Pat. No. 4,816,567)by substituting human complementarity determining regions withcorresponding rodent complementarity determining regions. Accordingly,such humanised antibodies are chimaeric antibodies, whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanised antibodies may be typically human antibodies inwhich some complementarity determining region residues and possibly someframework residues are substituted by residues from analogous sites inrodent antibodies.

Human antibodies can also be identified using various techniques knownin the art, including phage display libraries (see, for example,Hoogenboom & Winter, 1991, J. Mol. Diol. 227:381; Marks et al., 1991, J.Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies andCancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991. J. Immunol.147:86-95).

Once suitable antibodies are obtained, they may be tested for activity,for example by ELISA.

In an alternative embodiment of the first aspect of the invention, theagent comprises or consists of a non-immunoglobulin binding moiety, forexample as described in Skerra, Curr Opin Biotechnol. 2007 August;18(4):295-304.

In a further alternative embodiment, the agent comprises or consists ofan aptamer. For example, the agent may comprise or consist of a peptideaptamer or a nucleic acid aptamer (see Hoppe-Seyler & Butz, 2000, J MolMed. 78 (8): 426-30; Bunka D H & Stockley P G, 2006, Nat Rev Microbiol.4 (8): 588-96 and Drabovich et al., 2006, Anal Chem. 78 (9): 3171-8).

In a still further alternative embodiment, the agent comprises orconsists of a small chemical entity. Such entities with IL1RAP bindingproperties may be identified by screening commercial libraries of smallcompounds (for example, as available from ChemBridge Corporation, SanDiego, USA)

In addition to the binding moiety, the agents of the invention mayfurther comprise a moiety for increasing the in vivo half-life of theagent, such as but not limited to polyethylene glycol (PEG), human serumalbumin, glycosylation groups, fatty acids and dextran. Such furthermoieties may be conjugated or otherwise combined with the binding moietyusing methods well known in the art.

Likewise, it will be appreciated that the agents of the invention mayfurther comprise a cytotoxic moiety.

For example, the cytotoxic moiety may comprise or consist of aradioisotope, such as astatine-211, bismuth-212, bismuth-213,iodine-131, yttrium-90, lutetium-177, samarium-153 and palladium-109.

Alternatively, the cytotoxic moiety may comprise or consist of a toxin(such as saporin or calicheamicin).

In a further alternative, the cytotoxic moiety may comprise or consistof a chemotherapeutic agent (such as an antimetabolite).

Likewise, it will be appreciated that the agents of the invention mayfurther comprise a detectable moiety.

For example, the detectable moiety may comprise or consist of aradioisotope., such as technitium-99m, indium-111, gallium-67,gallium-68, arsenic-72, zirconium-89, iodine-12 or thallium-201.

Alternatively, the detectable moiety comprises or consists of aparamagnetic isotope, such as gadolinium-157, manganese-55,dysprosium-162, chromium-52 or iron-56.

Cytotoxic and detectable moieties may be conjugated or otherwisecombined with the binding moiety using methods well known in the art(for example, the existing immunoconjugate therapy, gemtuzumabozogamicin [tradename: Mylotarg®], comprises a monoclonal antibodylinked to the cytotoxin calicheamicin).

A third aspect of the invention provides a pharmaceutical compositioncomprising an effective amount of an agent as defined in relation to thefirst or second aspects of the invention together with a pharmaceuticalacceptable buffer, diluent, carrier, adjuvant or excipient.

Additional compounds may also be included in the compositions,including, chelating agents such as EDTA, citrate, EGTA or glutathione.

The pharmaceutical compositions may be prepared in a manner known in theart that is sufficiently storage stable and suitable for administrationto humans and animals. For example, the pharmaceutical compositions maybe lyophilised, e.g. through freeze drying, spray drying, spray cooling,or through use of particle formation from supercritical particleformation.

By “pharmaceutically acceptable” we mean a non-toxic material that doesnot decrease the effectiveness of the IL1RAP-binding activity of theagent of the invention. Such pharmaceutically acceptable buffers,carriers or excipients are well-known in the art (see Remington'sPharmaceutical Sciences, 18th edition, A. R Gennaro, Ed., MackPublishing Company (1990) and handbook of Pharmaceutical Excipients, 3rdedition, A. Kibbe, Ed., Pharmaceutical Press (2000), he disclosures ofwhich are incorporated herein by reference).

The term “buffer” is intended to mean an aqueous solution containing anacid-base mixture with the purpose of stabilising pH. Examples ofbuffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes,HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate,borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate,CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole,imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO andTES.

The term “diluent” is intended to mean an aqueous or non-aqueoussolution with the purpose of diluting the agent in the pharmaceuticalpreparation. The diluent may be one or more of saline, water,polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term “adjuvant” is intended to mean any compound added to theformulation to increase the biological effect of the agent of theinvention. The adjuvant may be one or more of zinc, copper or silversalts with different anions, for example, but not limited to fluoride,chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate,carbonate, lactate, glycolate, citrate, borate, tartrate, and acetatesof different acyl composition. The adjuvant may also be cationicpolymers such as cationic cellulose ethers, cationic cellulose esters,deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationicsynthetic polymers such as poly(vinyl imidazole), and cationicpolypeptides such as polyhistidine, polylysine, polyarginine, andpeptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids andminerals. Examples of carbohydrates include lactose, glucose, sucrose,mannitol, and cyclodextrines, which are added to the composition, e.g.,for facilitating lyophilisation. Examples of polymers are starch,cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose,alginates, carageenans, hyaluronic acid and derivatives thereof,polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone, all of different molecular weight, which are addedto the composition, e.g., for viscosity control, for achievingbioadhesion, or for protecting the lipid from chemical and proteolyticdegradation. Examples of lipids are fatty acids, phospholipids, mono-,di-, and triglycerides, ceramides, sphingolipids and glycolipids, all ofdifferent acyl chain length and saturation, egg lecithin, soy lecithin,hydrogenated egg and soy lecithin, which are added to the compositionfor reasons similar to those for polymers. Examples of minerals aretalc, magnesium oxide, zinc oxide and titanium oxide, which are added tothe composition to obtain benefits such as reduction of liquidaccumulation or advantageous pigment properties.

The agents of the invention may be formulated into any type ofpharmaceutical composition known in the art to be suitable for thedelivery thereof.

In one embodiment, the pharmaceutical compositions of the invention maybe in the form of a liposome, in which the agent is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids, which exist in aggregated forms as micelles,insoluble monolayers and liquid crystals. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Suitable lipids also include the lipids above modified bypoly(ethylene glycol) in the polar headgroup for prolonging bloodstreamcirculation time. Preparation of such liposomal formulations is can befound in for example U.S. Pat. No. 4,235,871, the disclosures of whichare incorporated herein by reference.

The pharmaceutical compositions of the invention may also be in the formof biodegradable microspheres. Aliphatic polyesters, such as polylacticacid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA)or poly(carprolactone) (PCL), and polyanhydrides have been widely usedas biodegradable polymers in the production of microspheres.Preparations of such microspheres can be found in U.S. Pat. No.5,851,451 and in EP 0 213 303, the disclosures of which are incorporatedherein by reference.

In a further embodiment, the pharmaceutical compositions of theinvention are provided in the form of polymer gels, where polymers suchas starch, cellulose ethers, cellulose carboxymethylcellulose,hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethylcellulose, alginates, carageenans, hyaluronic acid and derivativesthereof, polyacrylic acid, polyvinyl imidazole, polysulphonate,polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropyleneoxide copolymers, polyvinylalcohol/polyvinylacetate of different degreeof hydrolysis, and polyvinylpyrrolidone are used for thickening of thesolution containing the agent. The polymers may also comprise gelatin orcollagen.

Alternatively, the agents may simply be dissolved in saline, water,polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil),tragacanth gum, and/or various buffers.

It will be appreciated that the pharmaceutical compositions of theinvention may include ions and a defined pH for potentiation of actionof the active agent. Additionally, the compositions may be subjected toconventional pharmaceutical operations such as sterilisation and/or maycontain conventional adjuvants such as preservatives, stabilisers,wetting agents, emulsifiers, buffers, fillers, etc.

The pharmaceutical compositions according to the invention may beadministered via any suitable route known to those skilled in the art.Thus, possible routes of administration include parenteral (intravenous,subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar,buccal, oral, parenteral, vaginal and rectal. Also administration fromimplants is possible.

In one preferred embodiment, the pharmaceutical compositions areadministered parenterally, for example, intravenously,intracerebroventricularly, intraarticularly, intra-arterially,intraperitoneally, intrathecally, intraventricularly, intrasternally,intracranially, intramuscularly or subcutaneously, or they may beadministered by infusion techniques. They are conveniently used in theform of a sterile aqueous solution which may contain other substances,for example, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary. The preparation of suitableparenteral formulations under sterile conditions is readily accomplishedby standard pharmaceutical techniques well known to those skilled in theart.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Thus, the pharmaceutical compositions of the invention are particularlysuitable for parenteral, e.g. intravenous, administration.

Alternatively, the pharmaceutical compositions may be administeredintranasally or by inhalation (for example, in the form of an aerosolspray presentation from a pressurised container, pump, spray ornebuliser with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoro-methane,dichlorotetrafluoro-ethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane(HFA 227EA3), carbon dioxide or other suitable gas). In the case of apressurised aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. The pressurised container, pump,spray or nebuliser may contain a solution or suspension of the activepolypeptide, e.g. using a mixture of ethanol and the propellant as thesolvent, which may additionally contain a lubricant, e.g. sorbitantrioleate. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated to contain a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

The pharmaceutical compositions will be administered to a patient in apharmaceutically effective dose. A ‘therapeutically effective amount’,or ‘effective amount’, or ‘therapeutically effective’, as used herein,refers to that amount which provides a therapeutic effect for a givencondition and administration regimen. This is a predetermined quantityof active material calculated to produce a desired therapeutic effect inassociation with the required additive and diluent, i.e. a carrier oradministration vehicle. Further, it is intended to mean an amountsufficient to reduce and most preferably prevent, a clinicallysignificant deficit in the activity, function and response of the host.Alternatively, a therapeutically effective amount is sufficient to causean improvement in a clinically significant condition in a host. As isappreciated by those skilled in the art, the amount of a compound mayvary depending on its specific activity. Suitable dosage amounts maycontain a predetermined quantity of active composition calculated toproduce the desired therapeutic effect in association with the requireddiluent. In the methods and use for manufacture of compositions of theinvention, a therapeutically effective amount of the active component isprovided. A therapeutically effective amount can be determined by theordinary skilled medical or veterinary worker based on patientcharacteristics, such as age, weight, sex, condition, complications,other diseases, etc., as is well known in the art. The administration ofthe pharmaceutically effective dose can be carried out both by singleadministration in the form of an individual dose unit or else severalsmaller dose units and also by multiple administrations of subdivideddoses at specific intervals. Alternatively, the does may be provided asa continuous infusion over a prolonged period.

The polypeptides can be formulated at various concentrations, dependingon the efficacy/toxicity of the compound being used. Preferably, theformulation comprises the active agent at a concentration of between 0.1μM and 1 mM, more preferably between 1 μM and 500 μM, between 500 μM and1 mM, between 300 μM and 700 μM, between 1 μM and 100 μM, between 100 μMand 200 μM, between 200 μM and 300 μM, between 300 μM and 400 μM,between 400 μM and 500 μM and most prcfcrably about 500 μM.

It will be appreciated by persons skilled in the art that thepharmaceutical compositions of the invention may be administered aloneor in combination with other therapeutic agents used in the treatment ofa neoplastic hematologic disorder, such as inhibitors of tyrosine kinase(e.g. imatinib mesylate [Glivec®], dasatinib, nilotinib), omacetaxine,antimetabolites (e.g. cytarabine, hydroxyurea), alkylating agents,Interferon alpha-2b and/or steroids.

A fourth aspect of the invention provides a kit comprising an agent asdefined in relation to the first or second aspects of the invention or apharmaceutical composition according to the third aspect of theinvention.

A fifth aspect of the invention provides the use of an agent as definedin relation to the first or second aspects of the invention in thepreparation of a medicament for inducing cell death and/or inhibitingthe growth and/or proliferation of pathological stem cells and/orprogenitor cells associated with a neoplastic hematologic disorder,wherein the stem cells and/or progenitor cells express IL1RAP.

The agent may also be for use in inducing differentiation ofpathological stem and/or progenitor cells which express IL1RAP.

A related sixth aspect of the invention provides the use of an agent asdefined in relation to the first or second aspects of the invention inthe preparation of a diagnostic agent for detecting pathological stemcells and/or progenitor cells associated with a neoplastic hematologicdisorder, wherein the stem cells and/or progenitor cells express IL1RAP.

A related seventh aspect of the invention provides the use of an agentas defined in relation to the first or second aspects of the inventionfor detecting pathological stem cells and/or progenitor cells associatedwith a neoplastic hematologic disorder, wherein the stem cells and/orprogenitor cells express IL1RAP.

In one embodiment of the above use aspects of the invention, theneoplastic hematologic disorder is a leukemia.

In a further embodiment, the neoplastic hematologic disorder may beassociated with cells comprising a BCR/ABL1 fusion gene. For example,the pathological stem cells and/or progenitor cells may comprise aBCR/ABL1 fusion gene.

In a related embodiment, the neoplastic hematologic disorder may beassociated with cells comprising a Ph chromosome. For example, thepathological stem cells and/or progenitor cells comprise a Phchromosome. By “Ph chromosome” in this context we mean a specificchromosomal abnormality resulting from a reciprocal translocationbetween chromosome 9 and 22, specifically designated t(9;22)(q34;q11).An example of a neoplastic hematologic disorder associated with cellscomprising a Ph chromosome is chronic myeloid (or myelogenous) leukemia(CML).

However, it will be appreciated by persons skilled in the art that theagents of the invention may also be used in the treatment and/ordiagnosis of neoplastic hematologic disorders which are not associatedwith cells comprising a Philadelphia (Ph) chromosome (but neverthelessshow upregulation of IL1RAP). Such neoplastic hematologic disorderswhich are associated with cells which do not comprise a Ph chromosomeinclude the myelodysplastic syndromes (MDS) and myeloproliferativedisorders (MPD) such as polycythemia vera (PV), essential thrombocytosis(ET) and myelofibrosis (MF).

More specifically, the neoplastic hematologic disorder may be selectedfrom the group consisting of chronic myeloid leukemia (CML),myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS),acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

In one particularly preferred embodiment, the neoplastic hematologicdisorder is chronic myeloid leukemia (CML).

A eighth aspect of the invention provides a method for inducing celldeath and/or inhibiting the growth and/or proliferation of pathologicalstem cells and/or progenitor cells associated with a neoplastichematologic disorder in an individual, comprising the step ofadministering to the individual an effective amount of an agent asdefined in relation to the first or second aspects of the invention, ora pharmaceutical composition according to the third aspect of theinvention, wherein the stem cells and/or progenitor cells expressIL1RAP.

The method may also be for inducing differentiation of pathological stemand/or progenitor cells which express IL1RAP.

Thus the invention provides methods for the treatment of neoplastichematologic disorders. By ‘treatment’ we include both therapeutic andprophylactic treatment of the patient. The term ‘prophylactic’ is usedto encompass the use of a polypeptide or formulation described hereinwhich either prevents or reduces the likelihood of a neoplastichematologic disorder in a patient or subject.

A ninth aspect of the invention provides a method for detectingpathological stem cells and/or progenitor cells associated withneoplastic hematologic disorder in an individual, comprising the step ofadministering to the individual an effective amount of an agent asdefined in relation to the first or second aspects of the invention, ora pharmaceutical composition according to the third aspect of theinvention, wherein the stem cells and/or progenitor cells expressIL1RAP.

A tenth aspect of the invention provides an in vitro method fordiagnosing or prognosing a neoplastic hematologic disorder, the methodcomprising:

-   -   (a) providing a bone marrow or peripheral blood sample of        haematopoietic cells from an individual to be tested;    -   (b) isolating a subpopulation of CD34⁺, CD38⁻ cells from the        haematopoietic cells; and    -   (c) determining whether stem cells, contained within the CD34⁺,        CD38⁻ cells, express the cell surface markers IL1RAP.

wherein stem cells that exhibit the cell surface marker profile CD34⁺,CD38⁻ and IL1RAP⁺ exhibit are indicative of the individual having ordeveloping leukemia.

In one embodiment of the above method aspects of the invention, theneoplastic hematologic disorder is a leukemia.

In a further embodiment, the neoplastic hematologic disorder may beassociated with cells comprising a BCR/ABL1 fusion gene. For example,the pathological stem cells may comprise a BCR/ABL1 fusion gene.

In a related embodiment, the neoplastic hematologic disorder may beassociated with cells comprising a Ph chromosome. For example, thepathological stem cells comprise a Ph chromosome. By “Ph chromosome” inthis context we mean a specific chromosomal abnormality resulting from areciprocal translocation between chromosome 9 and 22, specificallydesignated t(9;22)(q34;q11). An example of a neoplastic hematologicdisorder associated with cells comprising a Ph chromosome is chronicmyeloid (or myelogenous) leukemia (CML).

However, it will be appreciated by persons skilled in the art that theagents of the invention may also be used in the treatment and/ordiagnosis of neoplastic hematologic disorders which are not associatedwith cells comprising a Philadelphia (Ph) chromosome (but neverthelessshow upregulation of IL1RAP). Such neoplastic hematologic disorderswhich are associated with cells which do not comprise a Ph chromosomeinclude the myelodysplastic syndromes (MDS) and myeloproliferativedisorders (MPD) such as polycythemia vera (PV), essential thrombocytosis(ET) and myelofibrosis (MF).

More specifically, the neoplastic hematologic disorder may be selectedfrom the group consisting of chronic myeloid leukemia (CML),myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS),acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

In one particularly preferred embodiment, the neoplastic hematologicdisorder is chronic myeloid leukemia (CML).

Preferred, non-limiting examples which embody certain aspects of theinvention will now be described, with reference to the followingfigures:

FIG. 1. P210 BCR/ABL1 Expression Induces IL1RAP Expression in Cord BloodCD34⁺ Cells

Flow cytometric analysis confirms that IL1RAP expression is induced uponretroviral P210 BCR/ABL1 expression in cord blood CD34⁺ cells, threedays post transduction. CD34⁺ GFP⁺ cells were gated according to thegates in the dot plots. The histogram shows the expression of IL1RAP fornegative control staining (white), MIG control (light gray) and MIG-P210(dark gray). The numbers in the dot plots show the percentage of cellswithin individual gates/quadrants. A representative experiment out ofthree is shown.

FIGS. 2A-C. IL1RAP is Upregulated in Primitive CML Cells

In FIG. 2A, FACS analysis on CD34⁺ cells from five CML patients and from2 normal bm samples. FACS dot plot showing gating for CD34⁺CD38⁺ orCD34⁺CD38⁻ cells in a representative CML patient.

FIG. 2B shows histograms of IL1RAP expression within CD34⁺CD38⁺ cells.

FIG. 2C shows histograms of IL1RAP expression within CD34⁺CD38⁻ cells.White represent control stained samples and gray represent IL1RAPstained samples. The sorting gates for CD34⁺CD38⁻IL1RAP⁻ andCD34⁺CD38⁻IL1RAP⁺ cells are outlined in the histograms. The numbers inthe dot plot and histograms show the percentage of cells withinindividual gates/quadrants.

FIG. 3. IL1RAP Expression Distinguishes Ph⁺ from Ph⁻ CML Cells withinthe CD34⁺CD38⁻ Cell Compartment

Flow-drop-FISH on CML CD34⁺CD38⁻IL1RAP⁻ and CD34⁺CD38⁻IL1RAP⁺ cells from5 CML patient samples revealed an almost complete separation betweenBCR/ABL1⁻ and BCR/ABL1⁺ cells, respectively. Black bars representBCR/ABL1 negative cells and white bars represent BCR/ABL1 positivecells. Outlined at the top of each bar is the number of Ph⁺ cells of thetotal nuclei scored.

FIGS. 4A-B. IL1RAP Expression Distinguishes Ph⁺ CML Stem Cells fromNormal HSC

FIG. 4A shows the number of LTC-CFC derived from CD34⁺CD38⁻IL1RAP⁻ andCD34⁺CD38⁻IL1RAP⁺ cells. Black bars represent IL1RAP⁻ cells and whitebars represent IL1RAP⁺ cells. Interphase FISH on LTC-CFC.

In FIG. 4B black bars represent BCR/ABL1 negative cells and white barsrepresent BCR/ABL1 positive cells. Qutlined at the top of each bar isthe number of Ph⁺ cells of the total nuclei scored.

FIGS. 5A-B. -Killing of a CML Cell Line by Antibody Targeting of IL1RAP

FIG. 5A shows histograms of IL1RAP expression on KU812 cells derivedfrom a CML patient and containing a Philadelphia chromosome, compared toexpression on KG-1 cells lacking a Philadelphia chromosome. White showcontrol stained samples and gray show KMT-1 stained samples.

FIG. 5B shows the leukemic cell line KG-1 was devoid of IL1RAPexpression, whereas KU812 express IL1RAP. As a consequence, low level ofantibody induced cell death was observed in KG-1, while a dose-dependentADCC effect was observed using KMT-1 on KU812 cells. As a control forunspecific ADCC effects, a rabbit IgG antibody was also used in theexperiments. The graph shows the average and standard deviation ofantibody induced cell death from three independent experiments.

FIGS. 6A-B. Killing of CML Stem Cells by Antibody Targeting of IL1RAP

FIG. 6A shows the use of KMT-1, normal bone marrow CD34+CD38− cellsstained negative for IL1RAP, whereas CML CD34+CD38+ and CD34+CD38− cellsexpressed IL1RAP. Histograms on CML-1 are shown from a representativeexperiment. White show control stained samples and gray show KMT-1stained samples.

FIG. 6B shows that in, line with the level of IL1RAP expression, noobvious ADCC effect was seen using normal bone marrow CD34+CD38− cells,whereas KMT-1 induced a strong dose-dependent ADCC effect in both CMLCD34+ and CD34+CD38− cells. As a control for unspecific ADCC effects, arabbit IgG antibody was also used in the experiments. The graph showsthe average and standard deviation of antibody induced cell death fromthree independent experiments using CML-1, CML-3, CML-4, and four normalbone marrow samples.

FIGS. 7A-C. IL1RAP is Expressed Also on Primary ALL and AML Stem Cells

In FIG. 7A, acute myeloid leukemia (AML) cells were received frompatients at diagnosis. IL1RAP expression on CD34+CD38− and CD34+CD38+cells from a representative AML patient is presented.

FIG. 7B shows that the AML cell line MONO-MAC-6 and the ALL cell lineREH express IL1RAP.

In FIG. 7C, acute lymphoid leukemia (ALL) cells were received frompatients at diagnosis. IL1RAP expression on CD34+CD38− and CD34+CD38+cells from a representative Ph+ ALL patient is presented. White showcontrol stained samples and gray show IL1RAP stained samples.

FIG. 8. Killing of AML and ALL Cell Lines by Antibody Targeting ofIL1RAP

FIG. 8 shows, in ADCC assay, a KMT-1 dose dependent cell death wasinduced in both the MONO-MAC-6 and the REH cell line, suggesting thatIL1RAP targeting antibodies may have a broader therapeutic window thanjust CML. As a control for unspecific ADCC effects, a rabbit IgGantibody was also used in the experiments. The graph shows the averageand standard deviation of antibody induced cell death from threeindependent experiments.

FIGS. 9A-B. Killing of AML and ALL Stem Cells by Antibody Targeting ofIL1RAP

FIG. 9A shows that a KMT-1 induced cell death was observed in bothprimary AML CD34+CD38. and FIG. 9B shows ALL CD34+CD38− cells,confirming that IL1RAP targeting antibodies also have a therapeuticeffect in AML and ALL with upregulation of IL1RAP on their cell surface.As a control for unspecific ADCC effects, a rabbit IgG antibody was alsoused in the experiments. The graph shows the specific antibody inducedcell death.

FIGS. 10A-B. IL1RAP is Expressed on Leukemic Stem Cells from MPD and MDSPatients.

FIG. 10A shows contour plots showing IL1RAP expression in CD34+CD38−cells of two MPD patients (MPD-1 and MPD-2), with and without the JAK2mutation.

FIG. 10B has histograms showing IL1RAP expression in an MDS patientprogressed into AML. White show control stained samples and gray shows asample stained with anti-IL1RAP antibodies.

EXAMPLE 1

IL1RAP is a Cell Surface Biomarker for Chronic Myeloid Leukemia StemCells

Summary

Therapeutic strategies for chronic myeloid leukemia (CML) aiming atachieving a permanent cure of the disorder, will require a fulleradication of the CML stem cells. The CML stem cells, sharing thecapacity to self-renew with normal hematopoietic stem cells (HSCs),represent a small population of leukemic cells that so far have beenindistinguishable from normal (HSCs) using cell surface markers. Onestrategy to target the CML stem cell would be to identify a cell surfacebiomarker for CML stem cells, to which future therapeutic antibodiescould be directed. In this study, we identified IL1RAP as commonlyupregulated both in primitive CML CD34+ cells and as a consequence ofectopic P210 BCR/ABL1 expression using global gene expression analyses.We further show that IL1RAP expression divides the rare CD34+CD38− cellpopulation, harboring both CML and normal HSCs, into two fractions; onehaving low/absent expression, the other having higher IL1RAP expression.After establishing a protocol, allowing detection of BCR/ABL1 by FISH insmall numbers of sorted cells, we observed that within the CMLCD34+CD38− cells; the IL1RAP+ cells were BCR/ABL1+, whereas IL1RAP−cells were almost exclusively BCR/ABL1−. By further performing long termculture-initiating cell (LTC-IC) assays on the two cell populations, wefound that candidate CML stem cells and normal HSC could beprospectively separated. This study thus identifies IL1RAP as the firstcell surface biomarker distinguishing CML stem cells from normal HSC andopens up new avenues for therapeutic and diagnostic strategies in CML aswell as in related disorders such as acute myeloid leukemia (AML), acutelymphoblastic leukemia (ALL), myeloproliferative disorders (MPDs) andmyelodysplastic syndrome (MDS).

Introduction

To identify a cell surface biomarker for CML stem cells, we performedglobal gene expression analyses and identified the interleukin 1receptor accessory protein (IL1RAP) as the top candidate, beingupregulated both in primitive CML patient cells and as a consequence ofectopic P210 BCR/ABL1 expression. Upon development of an assay fordetecting BCR/ABL1 in low numbers of sorted cells, we show that theIL1RAP expression enables prospective separation of primitive leukemicand normal cells. Through long-term culturing-initiating cell assays, wefurther show that IL1RAP is a cell surface biomarker for CML stem cells,for the first time allowing prospective separation of CML stem cellsfrom normal HSC.

Material and Methods

Collection of CML Patient Cells

Isolation and Transduction of Cord Blood CD34⁺ Cells

Blood and occasionally bone marrow samples from CML patients wereobtained at diagnosis before treatment was initiated after informedconsent according to a protocol approved by the local ethical board.Samples were received both from the Department of Hematology at LundUniversity Hospital, Sweden and from Rigshospitalet, Copenhagen,Denmark. Mononuclear cells (MNCs) were separated using Lymphoprep™(Axis-Shield PoC AS, Oslo, Norway) according to the manufacturer'sinstructions and CD34⁺ cells were enriched using the CD34⁺ cellisolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) aspreviously described²², on a regular basis, this yielded a purity ofCD34⁺ cells above 95%. A subfraction of mononuclear cells was viablystored in liquid nitrogen before antibody staining was initiated. CD34⁺cells were split in two fractions; one fraction was washed in PBS andresuspended in Trizol and frozen in −80C, whereas the other fraction wasfrozen in liquid nitrogen. As reference samples, bone marrow samplesfrom healthy volunteers were obtained after informed consent at the LundUniversity Hospital, followed by CD34-cell isolation as described above.

Microarray Analysis

Microarray analysis was performed using oligonucleotide slides from theSwegene DNA Microarray Resource Center at Lund University, Sweden.Hybridizationss were performed using the Pronto Universal Hybridizationkit (Corning Inc, Corning, N.Y.). The RNA isolation and microarrayanalysis was performed essentially as previously described²³. Datavisualization was performed using the software Qlucore Omics Explorer2.0 (Qlucore, Lund, Sweden).

Flow Cytometric Analysis

Flow cytometric analyses were performed in a FACS Canto and flowcytometric cell sorting was done in a FACS Aria (both from BD). Prior tocell staining, CD34⁺ cells were thawed according to standard proceduresand washed once in PBS containing 2% FCS (washing medium).Biotin-labeled goat anti-human IL1RAP polyclonal antibody (batch 667,R&D Systems, Abingdon, UK) was used at a 1:100 dilution for staining thecells for 30 min on ice. Subsequently, the cells were washed andPE-conjugated streptavidin was used at a 1:200 dilution for 30 min. TheAPC-conjugated anti-CD34 and FITC-conjugated anti-CD38 monoclonalantibodies were used for co-staining (except IL1RAP all antibodies usedwere purchased from Beckton-Dickinson Immunocytometry Systems, MountainView, Calif.). Before cell sorting, cells were washed twice to avoidunspecific binding of PE-conjugated streptavidin. Isotype matchingcontrol antibodies were used as negative controls.

Cell Sorting and Interphase FISH

Glass slides were treated with 0.01% poly L-lysine (Sigma-Aldrich,Stockholm, Sweden) for two hours while kept in a moist chamber, washedonce in water, and dried on a hot plate at 37° C. until dry.Subsequently, a hydrophobic pen (Daido Sangyo Co., Ltd. Tokyo, Japan)was used to draw circles with a 96-well tissue culture plate astemplate. Prior to cell sorting, but after at least two hours drying inroom temperature, 25 μL PBS was applied to the rings to form drops.During cell sorting, 30 to 3000 cells were sorted simultaneouslydirectly into two drops. To allow attachment of the cells to the surfaceand to avoid drying of the drops, slides were maintained in a moistchamber on ice for 30 min before cells were fixed in methanol:aceticacid (3:1) for 10 min. Subsequently, slides were incubated in a 70° C.oven over night, followed by FISH. Dual color probes for BCR/ABL1(Abbot, Wiesbaden, Germany) were used.

Long Term Culture-Initiating Cells (LTC-IC)

M₂10B₄ stroma cells were cultured in RPMI-1640 medium supplemented with10% FCS as previously described^(24, 26). Two days prior to cellsorting, stroma cells were seeded into wells of a 96-well plate atdensity of 50,000 cells per mL in 200 82 L Myelocult medium (Stem CellTechnologies, Vancouver, Canada) containing 10⁻⁶ M Hydrocortisone(Sigma-Aldrich, Stockholm, Sweden). Twenty-four hours before cellsorting, stroma cell were irradiated with 1000 Rad. During cell sorting,100-500 cells were sorted directly into the stroma-precoated wells induplicate and 100 μL medium was exchanged 3 h later. Once per week, theexchange of 100 μL culture medium was repeated. After 5-6 weeks culture,cells were washed and plated in methylcellusose medium (MethoCultH44435; Stem Cell Technologies) in a 24-well plate. Two weeks later, thenumber of colonies was scored. Colonies from individual wells werepooled, washed, applied to PBS drops on slides, and followed by FISHanalysis as described above.

P210 BCR/ABL1 Expression in Cord Blood CD34⁺ Cells

Umbilical cord blood samples were collected from normal deliveries afterobtaining informed consent according to a protocol approved by the localethical board. CD34⁺ cells were enriched as previously described²²,yielding a purity of CD34⁺ cells above 95%. The RD114 pseudotypedMSCV-IRES-GFP (MIG) and MIG-P210 viral vectors were used in thisstudy²³. CD34⁺ cells were cultured and transduced in SFMM medium (StemCell Technology, Vancouver, Canada) supplemented with thrombopoietin(TPO; 50 ng/mL), stem cell factor (SCF; 100 ng/mL), and Flt-3-ligand(FL; 100 ng/mL) as previously described²³.

Results and Discussion

Global Gene Expression Analysis Identifies IL1RAP as Upregulated on CMLCD34⁺ Cells

Much effort has been put into investigations aimed at identifying a cellsurface biomarker for Ph⁺ CML stem cells (reviewed by C Eaves¹⁴).Leukemic and normal cells can rather easily be identifiedretrospectively in CML following detection of the leukemia specificBCR/ABL1 fusion gene by FISH, making it an ideal disorder for evaluatingattempts to prospectively separate leukemic and normal cells. However,so far, no cell surface marker has been identified that allowsprospective separation of CML stem cells from normal HSC. Global geneexpression analyses have proven to be a powerful strategy in searchingfor new HSC markers such as the SLAM receptors distinguishinghematopoietic stem and progenitor cells¹⁵. To search for upregulatedgenes encoding candidate cell surface biomarkers for CML stem cells, thetranscriptional profiles of CD34⁺ cells from 11 CML patient samples and5 normal bone marrow (bm) samples were compared. The identifiedupregulated genes in CML were matched to the Gene Ontology (GO) category“integral to plasma membrane” that had been manually curated to includeall known CD molecules (see Material and Methods for details). In total,13 upregulated genes in CML CD34⁺ cells matched to the integral toplasma membrane gene category (data not shown). To further link theupregulated genes more directly to P210 BCR/ABL1 expression, we inparallel generated a list of upregulated genes as a consequence of P210BCR/ABL1 expression in cord blood CD34⁺ cells. This analysis resulted in23 upregulated genes matching to the same GO category gene list (datanot shown). Interestingly, only one gene, the Interleukin 1 receptoraccessory protein (IL1RAP), showed a strong upregulation both in CD34⁺CML cells and in cord blood CD34⁺ cells as a consequence of P210BCR/ABL1 expression. The findings that IL1RAP was present on both genelists suggest that its upregulation on primitive CML cells is closelycoupled to the P210 BCR/ABL1 expression and indicate that IL1RAP is anovel leukemia-associated antigen on primitive CML cells.

IL1RAP is Upregulated on CD34⁺CD38⁻ Cells from CML Patients and isInduced as a Consequence of Ectopic P210 BCR/ABL1 Expression

IL1RAP is a member of the Toll-like receptor superfamily and is awell-known co-receptor to Interleukin 1 receptor type 1 (IL-1R1)¹⁶.IL1RAP is thus crucial in mediating the effect of the pro-inflammatorycytokine IL-1, but it is also involved in mediating the signal of IL-33,a cytokine that activates T-cells and mast cells through binding itsreceptor ST2, which subsequently dimerizes with IL1RAP¹⁷. IL-1R1activation has previously been shown to stimulate colony growth ofinterferon sensitive CML cells¹⁸, however, IL1RAP has to our knowledgenot previously been linked directly to CML.

As P210 BCR/ABL1 is present in CML cells as a hallmark of the disease,ideally, a reliable cell surface biomarker in CML, should be directlycoupled to the presence and expression of P210 BCR/ABL1. In agreementwith the microarray data, IL1RAP expression was indeed upregulated onthe cell surface on CB CD34⁺ cells following retroviral P210 BCR/ABL1expression (FIG. 1). This suggests that P210 BCR/ABL1 regulates IL1RAPexpression, either directly or through an indirect effect, strengtheningits candidature as a CML biomarker.

We next investigated the cell surface IL1RAP expression on CMLCD34⁺CD38⁺ cells, representing the majority and more mature CD34⁺ cells.In this cell population, an upregulation of IL1RAP was observed comparedto the expression in corresponding normal bm cells (FIGS. 2A, B). Thenormal CD34⁺CD38⁺ cells displayed a lower IL1RAP expression thatpartially overlapped with the expression on CML cells. We then turned tothe CD34⁺CD38⁻ cell compartment of normal cells, containing the HSCs. Inagreement with a previous study, this population displayed a low/absentIL1RAP expression (FIG. 2C)¹⁹. Strikingly, the CD34⁺CD38⁻ cells from CMLpatients, harboring both Ph⁺ CML stem cells and normal HSCs were dividedinto two populations; one having low/absent IL1RAP expression, the otherhaving higher IL1RAP expression (FIG. 2C). In the peripheral blood (PB)of five CML patients, the IL1RAP positive cell fraction constitutedbetween 75% and 95% of the CD34⁺CD38⁻ cells (n=5). Based on thesefindings, we speculated that the IL1RAP expression might distinguishnormal and leukemic cells within the CD34⁺CD38 ⁻ cell compartment inCML. As all CML stem cells and normal HSC exclusively are found withinthe CD34⁺CD38⁻ cells, such separation between normal and leukemic cells,would allow a prospective separation of CML stem cells from normal HSC.

Flow-Drop-FISH Shows that IL1RAP Expression Separates Normal andLeukemic Cells within CML CD34⁺CD38⁻ Cells

To test whether the IL1RAP expression distinguishes normal (Ph⁻) andleukemic (Ph+) cells within the CML CD34⁺CD38⁻ cell compartment, weestablished a new protocol for doing fluorescent in situ hybridization(FISH) on small numbers of sorted cells (see Material and Methods). Thefirst steps in this protocol is partly based on a method for sortingcells into drops on slides followed by single cell immuno-staining²⁰. Byapplying this new protocol involving cell sorting directly into drops onslides followed by FISH, hereafter referred to as Flow-drop-FISH, wesorted as few as 30 cells into a drop, from which 15 nuclei weresuccessfully scored by FISH (CML-5, FIG. 3). Interestingly, we found byFlow-drop-FISH that the CML CD34⁺CD38⁻IL1RAP⁺ cells were BCR/ABL1⁺,whereas CML CD34⁺CD38⁻ IL1RAP⁻ cells were almost exclusively Ph⁻ (n=5,FIG. 3). These data show that IL1RAP expression separates leukemic andnormal cells within the CML CD34⁺CD38⁻ cell compartment, indicating thatCML stem cells and normal HSC can be prospectively separated

CML Stem Cells are CD34⁺CD38⁻IL1RAP⁺ whereas Normal HSC areCD34⁺CD38⁻IL1RAP^(−/low)

Studies on chronic phase CML stem cells has so far relayed on access torare CML patients in which the stem cells compartment have beendominated by leukemic cells following long-term assays¹⁴. As CML stemcells generally show poor engraftment in immuno-deficient mice, thelong-term culture initiating cell (LTC-IC)-assay is widely used as asurrogate assay for detection of candidate CML stem cells. To testwhether CML CD34⁺CD38⁻IL1RAP⁺ and CD34⁺CD38⁻IL1RAP^(−/low) uniquelycontain candidate CML stem cells and normal HSC, respectively, we testedthe two cell populations in the LTC-IC assay. For bone marrow CD34⁺cells from normal controls, long term culture—colony forming cells(LTC-CFC) were found at an >100-fold higher frequency amongCD34⁺CD38⁻IL1RAP⁻ cells compared to CD34⁺CD38⁻IL1RAP⁺ cells (FIG. 4A,n=2), indicating that normal CD34⁺CD38⁻IL1RAP⁻ are hierarchically on topof CD34⁺CD38⁻IL1RAP⁺ cells. In CML, we observed on average a 3.6-foldhigher frequency of LTC-CFC within the CD34⁺CD38⁻IL1RAP⁻ cells comparedto the CD34⁺CD38⁻IL1RAP⁺ cells (n=5, FIG. 4A), suggesting that CMLCD34⁺CD38⁻IL1RAP⁻ cells are more enriched for primitive cells.Importantly, although a higher number of LTC-IC were found amongCD34⁺CD38⁻IL1RAP⁻ cells than within CD34⁺CD38⁻IL1RAP⁺ cells from bothCML patient samples and from normal controls, FISH on CML LTC-coloniesrevealed an almost complete discrimination between Ph⁻ and Ph⁺ cells inthe two groups (FIG. 4B). CML LTC-colonies derived fromCD34⁺CD38⁻IL1RAP⁻ cells were almost exclusively Ph⁻, whereasCD34⁺CD38⁻IL1RAP⁺ were almost exclusively Ph⁺. These data suggest thatIL1RAP is a novel cell surface biomarker that can be used to separateCML stem cells from normal HSC.

Herein, we identified through global gene expression analysis a novelcell surface antigen, IL1RAP, that following challenging in multipleassays fulfilled the criteria for being a novel cell surface biomarkerfor Ph⁺ CML stem cells. Based on this discovery, future directedtherapies in CML could be designed to target the CML stem cells whilepreserving normal HSC by using a therapeutic antibody directed towardsIL1RAP. In addition, an antibody cocktail containing anti-CD34,anti-CD38 and anti-IL1RAP antibodies can be used for diagnostic purposesand for follow-up studies of CML patients under different treatments.Importantly, a prospective separation of normal and CML stem cells willenable future mechanistic studies of these two cell populations.Moreover, we here also show that Flow-drop-FISH could serve as a usefulmethod in characterizing genetic aberrations in small numbers of sortedcells, such as leukemic stem cells, a cell type that has been purifiedto increasingly smaller and purer cell populations²¹. For futurestudies, this method would for example allow detection of geneticalaberrations in various small leukemic stem and progenitor cellpopulations, findings that are likely to provide novel insights intowhich orders the various aberrations have been acquired, key knowledgeto understand leukemogenesis. In addition, Flow-drop-FISH could be usedto monitor therapeutic effects on leukemic stem cells during treatment.Importantly, we here identified by using Flow-drop-FISH that IL1RAP isthe first cell surface biomarker that distinguishes CML stem cells fromnormal HSCs, a finding that opens up new therapeutic opportunities forCML and other neoplastic hematologic disorders associated withupregulation of IL1RAP on stem cells and/or progenitor cells.

REFERENCES

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Targeting        of CD44 eradicates human acute myeloid leukemic stem cells. Nat        Med. 2006; 12:1167-1174.    -   7. Tavor S, Petit I, Porozov S, et al. CXCR4 regulates migration        and development of human acute myelogenous leukemia stem cells        in transplanted NOD/SCID mice. Cancer Res. 2004; 64:2817-2824.    -   8. Jin L, Lee E M, Ramshaw H S, et al. Monoclonal        antibody-mediated targeting of CD123, IL-3 receptor alpha chain,        eliminates human acute myeloid leukemic stem cells. Cell Stem        Cell. 2009; 5:31-42.    -   9. Majeti R, Chao M P, Alizadeh A A, et al. CD47 is an adverse        prognostic factor and therapeutic antibody target on human acute        myeloid leukemia stem cells. Cell. 2009; 138:286-299.    -   10. Hosen N, Park C Y, Tatsumi N, et al. CD96 is a leukemic stem        cell-specific marker in human acute myeloid leukemia. Proc Natl        Acad Sci USA. 2007; 104:11008-11013.    -   11. van Rhenen A, van Dongen G A, Kelder A, et al. The novel AML        stem cell associated antigen CLL-1 aids in discrimination        between normal and leukemic stem cells. Blood. 2007;        110:2659-2666.    -   12. Eisterer W, Jiang X, Christ O, et al. Different subsets of        primary chronic myeloid leukemia stem cells engraft        immunodeficient mice and produce a model of the human disease.        Leukemia. 2005; 19:435-441.    -   13. Bhatia M, Wang J C, Kapp U, Bonnet D, Dick J E. Purification        of primitive human hematopoietic cells capable of repopulating        immune-deficient mice. Proc Natl Acad Sci USA. 1997;        94:5320-5325.    -   14. Jiang X, Zhao Y, Forrest D, Smith C, Eaves A, Eaves C. Stem        cell biomarkers in chronic myeloid leukemia. Dis Markers. 2008;        24:201-216.    -   15. Kiel M J, Yilmaz O H, Iwashita T, Terhorst C, Morrison S J.        SLAM family receptors distinguish hematopoietic stem and        progenitor cells and reveal endothelial niches for stem cells.        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Adult mouse        hematopoietic stem cells: purification and single-cell assays.        Nat Protoc. 2006; 1:2979-2987.    -   21. Dick J E. Stem cell concepts renew cancer research. Blood.        2008; 112:4793-4807.    -   22. Nilsson M, Karlsson S, Fan X. Functionally distinct        subpopulations of cord blood CD34+ cells are transduced by        adenoviral vectors with serotype 5 or 35 tropism. Mol Ther.        2004; 9:377-388.    -   23. Jaras M, Johnels P, Agerstam H, et al. Expression of P190        and P210 BCR/ABL1 in normal human CD34(+) cells induces similar        gene expression profiles and results in a STAT5-dependent        expansion of the erythroid lineage. Exp Hematol. 2009;        37:367-375.    -   24. Hogge D E, Lansdorp P M, Reid D, Gerhard B, Eaves C J.        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EXAMPLE 2

Antibody-Targeting of IL1RAP on Leukemia Stem and Progenitor Cells CauseAntibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

Summary

Therapeutic strategies for leukemias aimed at achieving a permanent curewill require a full eradication of the leukemia stem cells. The leukemiastem cells, representing a small population of leukemic cells, have sofar have been indistinguishable from normal hematopoietic stem cells(HSCs) using cell surface markers. A new concept for targeting leukemiastem cells would be to identify a cell surface biomarker for leukemiastem cells, to which future therapeutic antibodies could be directed(see Example 1).

In this study, we generate an anti-IL1RAP antibody and provide proof ofconcept that anti-IL1RAP antibodies targeting chronic myeloid leukemia(CML) stem cells, Acute myeloid leukaemia (AML) stem cells, and Acutelymphoblastic leukaemia (ALL) stem cells can be used to induceantibody-dependent-cell-mediated cytotoxicity (ADCC), whereas nocytotoxic effect was observed on normal HSC. Furthermore, we demonstratea dose-dependent IL1RAP targeting ADCC in the IL1RAP positive cell linesKU812 (CML), MONO-MAC-6 (acute myeloid leukemia; AML) and REH (acutelymphoblastic cell line; ALL). We also demonstrate that MDS and MPD stemcells have increased IL1RAP expression, indicative that futuretherapeutic anti-IL1RAP targeting antibodies will be effective also inthese disorders.

This study thus opens up for a novel therapeutic opportunity in CML,AML, ALL, MDS, and MPD by antibody targeting of IL1RAP on leukemic stemcells.

Materials and Methods

Generation of KMT-1; A Polyclonal Rabbit Anti-Human IL1RAP Antibody

Rabbits were immunized with the extracellular domain of IL1RAP. Serumfrom rabbits were purified according to standard procedures, except thatan additional step was added, in which antibodies binding to theimmunoglobulin domain, present on the immunizing protein for increasedhalf-life, was discarded through binding to immunoglobulin loadedcolumns. Purified antibodies were confirmed in ELISA to bind theextracellular domain of IL1RAP and to be devoid of antibodies bindingthe human immunoglobulin domain. When used in flow cytometry, aPE-conjugated goat anti-rabbit IgG antibody was used as secondaryreagent.

ADCC Assay

The ADCC assay was based on a protocol previously described¹. In brief,target cells were labelled with PKH26 (Sigma-Aldrich, St Louis, Mo.)according to manufacturer's instructions and either cells were putdirectly into wells of a 96-well plate, or seeded into the wellsfollowing sorting of CD34⁺CD38⁻ cells. The KU812 and KG-1 cell lines andprimary CD34⁺ cells were seeded at 10,000 cells per well, whereasprimary CD34⁺CD38⁻ cells were seeded at 2,000-3,000 cells per well.Subsequently, antibodies were added to wells in different concentrationsand incubated for 20 min before 100,000 NK-effector cells were added toeach well. NK-cells were extracted from healthy volunteers afterinformed consent by using a NK-cell negative cell isolation kitaccording to manufacturer's instructions (Miltenyi Biotech, BergischGladbach, Germany). Rabbit IgG antibodies purified from a non-immunizedrabbit was used as control antibody in the experiments (R&D SystemsAbingdon, UK). 7-AAD positive cells for detection of cell death weremeasured using a FACS CANTO flow cytometer (BD). The average andstandard deviation of antibody induced cell death was calculatedaccording to the following equation: (Percentage 7-AAD+ cells at definedantibody concentration−Percentage 7-AAD+ cells withoutantibody)/(0.01×Percentage 7-AAD− cells without antibody) from at leastthree independent experiments (except FIG. 9; 1 experiment only).

Samples from eleven AML patients and two Ph+ ALL patients were receivedfrom Lund University hospital and the expression of IL1RAP was analyzedin the CD34⁺CD38⁺ and CD34⁺CD38⁻ cell populations using the samesettings as for the analysis of CML cells. The AML cell line MONO-MAC-6and the ALL cell line REH were also tested in ADCC assays using the samesetup as the for the KG-1 and KU812 cell lines.

Results

Antibody-Targeting of IL1RAP on CML Stem and Progenitor Cells but Alsoon a CML Cell Line Directs NK-Cells to ADCC

Antibody-dependent-cell-mediated cytotoxicity (ADCC) is a conservedmechanism of the innate immune system, through which several therapeuticantibodies, such as Rituximab directed against CD20, are believed to atleast partially exert their therapeutic effect². To test whether ADCCcould be achieved using IL1RAP as a target, we generated a polyclonalrabbit anti-human IL1RAP antibody hereafter referred to as KMT-1, as theFc domain of rabbit antibodies in contrast goat antibodies arerecognized by cells of the human immune system.

As expected, low levels of ADCC were observed in the IL1RAP negative/lowleukemia cell line KG-1, even at high KMT-1 concentrations (FIGS. 5A,B). In contrast, in the CML cell line KU812 expressing IL1RAP, a naturalkiller (NK)-cell mediated ADCC was observed in the presence of KMT-1(FIGS. 5A, B), demonstrating that KMT-1 has the potential to induce ADCCby recruiting cytotoxic immune cells to IL1RAP+ target cells.

On primary cells from CML patients and from normal controls, KMT-1showed a slightly weaker, but similar staining pattern as the previouslyused polyclonal goat antihuman IL1RAP antibody (Example 1, FIG. 6A).Immature cells from CML-1, CML-3 and CML-4 (no more cells remained fromCML-2 and CML-5) were tested in ADCC assays in parallel to cells fromhealthy control samples. In CML CD34⁺ cells, the binding of KMT-1resulted in ADCC at higher levels than in normal CD34⁺ control cells,correlating to the expression level of IL1RAP, in particular at lowerantibody concentrations (FIG. 6B). More strikingly, among the stem cellenriched CD34⁺CD38⁻ cells, KMT-1 did not induce ADCC of normalCD34⁺CD38⁻ cells, whereas a clear dose dependent ADCC effect wasobserved in CML CD34⁺CD38⁻ cells (FIG. 6B), again showing strongcorrelation to the expression pattern of ILA RAP on these cell types.

Antibodies Targeting IL1RAP on AML and ALL Cells Direct NK-Cells to ADCC

IL1RAP expression was observed in AML CD34⁺CD38⁻ cells in 9 out of 11tested samples (FIG. 7A). In the CD34⁺CD38⁺ cell population, a similarIL1RAP expression pattern was observed (FIG. 7A). In addition, IL1RAPwas expressed in the AML cell line MONO-MAC-6 and the ALL cell line REH(FIG. 7B). IL1RAP expression was also observed in Ph+ ALL CD34⁺CD38⁻cells in 2 out of 2 tested samples (FIG. 7C). Using IL1RAP as target,the MONOMAC-6 and REH cell lines were also tested in ADCC assays. Inboth these cell lines, a dose dependent IL1RAP targeting ADCC effect wasobserved (FIG. 8), demonstrating that therapeutic anti-IL1RAP targetingantibodies have a broader application than just CML.

We also performed ADCC experiments on primary AML and ALL CD34+CD38−cells and demonstrated proof of principle that also in these disorders,an increased cell death could be achieved using KMT-1 (FIG. 9).

In addition, CD34+CD38− cells from one MDS patient at progression intoAML and two MPD patients (one of them JAK2 mutation+) were stained withan IL1RAP targeting antibody. An increased IL1RAP expression wasobserved in comparison to normal bone marrow CD34+CD38−cells (FIG. 10,FIG. 2C).

Discussion

In the present study, we have identified IL1RAP as the first cellsurface biomarker that distinguishes candidate CML stem cells fromnormal HSCs and used this knowledge to induce an antibody-dependent cellkilling of CML stem cells. Further, we identified IL1RAP as upregulatedon AML stem cells, ALL stem cells, MPD stem cells and MDS stem cells andshowed that both AML and ALL stem cells can be killed using anIL1RAP-targeting antibody, whereas normal stem cells were unaffected.Based on the finding that CML, ALL and AML stem cells can be killed byIL1RAP targeting antibodies, it is expected that also MPD and MDS stemcell would be killed in the ADCC assay. These findings opens up a newconcept for treatments of leukemia patients by direct targeting of theleukemia stem cells, a concept that is distinct from the tyrosine kinaseinhibitors currently used, which preferentially target cells downstreamof the CML stem cells^(3, 4).

The reason why CML stem cells are resistant to drugs such as Glivec ispartially unclear, but factors that may contribute are features such asquiescence and relatively high level of BCR/ABL1 expression, but alsocombinatorial expression of specific membrane transporter proteins inthese cells^(3, 5, 6). Given these features of the CML stem cells, it ishighly desirable to find novel treatment approaches to ultimatelyeradicate the CML stem cells. An antibody-based therapy directlytargeting CML stem cells would serve in such a strategy as theantibodies mode of action is independent of the known resistantmechanisms causing CML stem cells to be unresponsive to kinase inhibitortreatments. The major limitations for such developments have been thecomplete lack of a cell surface receptor distinguishing CML Ph+ fromnormal, healthy (Ph−) stem cells. We herein identified IL1RAP as such atarget from global gene expression analyses and importantly linked itsexpression to BCR/ABL1 expression (see Example 1 above).

Importantly, by generation of an antibody targeting IL1RAP, we here, forthe first time, provide proof of concept that candidate CML stem cellscan be targeted while preserving normal HSC. Importantly, as theantibodies mode of action in ADCC is to direct immunological cells totarget cell killing, the therapeutic mechanisms is independent of theknown mechanisms causing kinase inhibitor resistance in CML usingcurrent treatments. Hence, antibody targeting of CML stem cells has thecapacity to eradicate CML stem cells, either alone or in combinationwith current regimens, ultimately leading to a permanent cure for CMLpatients.

Interestingly, we also observed that IL1RAP targeting antibodies cancause ADCC of AML stem cells; the most common type of acute leukemiaamong adults having a poor prognosis, and also ALL stem cells; the mostcommon type of childhood leukemia. Collectively, the finding of IL1RAPexpression on leukemic stem cells having a CD34⁺CD38⁻ immuno-phenotypein CML, AML, ALL, MDS, and MPD, and the ADCC experiments demonstratingcell killing in an IL1RAP dependent manner, indicates that thesedisorders can be treated with anti-IL1RAP therapeutic antibodies.

In the ADCC experiments presented herein, a polyclonal anti-human IL1RAPantibody was used (which is essentially a mixture of several differentmonoclonal antibodies). However, it will be appreciated by personsskilled in the art that individual monoclonal antibodies targetingIL1RAP can also be identified which have ADCC potential.

REFERENCES

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1-92. (canceled)
 93. A method of treatment of leukemia, the methodcomprising administering a therapeutically effective amount of anantibody with specificity for an extracellular domain of theinterleukin-1 receptor accessory protein (IL1RAP), wherein the antibodyinhibits IL1RAP binding activity associated signaling on pathologicalstem cells and/or progenitor cells expressing IL1RAP.
 94. The methodaccording to claim 93, wherein the pathological stem cells and/orprogenitor cells comprise a BCR/ABL1 fusion gene.
 95. The methodaccording to claim 93, wherein the leukemia is selected from the groupconsisting of chronic myeloid leukemia (CML), myeloproliferativedisorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblasticleukemia (ALL) and acute myeloid leukemia (AML).
 96. The methodaccording to claim 93, wherein the leukemia is CML.
 97. The methodaccording to claim 93, wherein the leukemia is MDP.
 98. The methodaccording to claim 93, wherein the leukemia is MDS.
 99. The methodaccording to claim 93, wherein the leukemia is ALL.
 100. The methodaccording to claim 93, wherein the leukemia is AML.
 101. The methodaccording to claim 93, wherein the pathological stem cells and/orprogenitor cells comprise a Ph chromosome.
 102. The method according toclaim 93, wherein the antibody blocks binding of one or moreco-receptors to IL1RAP.
 103. The method according to claim 102, whereinthe one or more co-receptors are selected from the group consisting ofIL1R1, ST2, C-KIT and IL1RL2.
 104. The method according to claim 93,wherein the antibody induces apoptosis of the stem cells and/orprogenitor cells.
 105. The method according to claim 93, wherein theantibody further comprises a cytotoxic moiety.
 106. The method accordingto claim 93, wherein the antibody blocks binding of IL1R1.
 107. A methodfor inducing cell death and/or inhibiting the growth and/orproliferation of pathological stem cells and/or progenitor cellsassociated with a neoplastic hematologic disorder in an individual,comprising the step of administering to the individual an effectiveamount of an agent comprising a binding moiety with specificity forinterleukin-1 receptor accessory protein (IL1RAP), wherein the cellsexpress IL1RAP.
 108. The method according to claim 107, wherein theagent is an antibody with specificity for an extracellular domain of theinterleukin-1 receptor accessory protein (IL1RAP).
 109. The methodaccording to claim 107, wherein the agent inhibits IL1RAP bindingactivity associated signaling on pathological stem cells and/orprogenitor cells expressing IL1RAP.
 110. The method according to claim107, wherein the agent blocks binding of one or more co-receptors toIL1RAP.
 111. A method for detecting pathological stem cells and/orprogenitor cells associated with neoplastic hematologic disorder in anindividual, comprising the step of administering to the individual aneffective amount of an agent comprising a binding moiety withspecificity for interleukin-1 receptor accessory protein (IL1RAP),wherein the cells express IL1RAP.